We are studying the effects of the cytokines IL-1, IL-6 and tumor necrosis factor alone and in combination to determine synergy, cell growth inhibition, and regulation of estradiol stimulated metabolism of breast cancer cells. We found that all three cytokines inhibit cell growth in vitro with the following efficacy: TNF more IL-1 more IL-6. IL-1 in combination with IL-6 causes greater inhibition and greater antagonism of E2 stimulation of growth of ER+ cells MCF-7 and T47D than either IL-1 or IL-6 alone. Growth inhibition/antagonism by this combination is dose dependent for both IL-1 and IL-6, and estradiol. Both IL-1 and IL-6 down-regulate the estrogen receptor. The combination has a greater effect than either IL-1 or IL-6 alone. TNF down-regulates the ER and upregulates the PR without altering steady-state levels of ER or PR mRNA. TNF increases insulin-like growth factor secretion, which may explain PR upregulation. IL-1 stimulates secretion of TGFBeta, which may explain IL-1 growth inhibition. Stimulation is dose-dependent for IL-1 and time-dependent. The combination of IL-1 and IL-6 produces a greater increase in TGFBeta secretion than IL-1 alone. Estradiol antagonizes IL-1 stimulation of TGFBeta secretion in a dose-dependent manner. IL-1 stimulates production of TNF mRNA in breast cancer cells. IL-1 induces the 26 kD membrane bound form of TNF protein, which is confirmed by Western blot analysis. IL-1 does not induce secretion of TNF. We are currently studying the effect of IL-1 plus/minus IL-6, and TNF in vivo on breast cancer cell growth and metabolism, and the kinetics, cellular distribution, and modulation of estradiol regulated metabolism of IL-1 induced TGFBeta in vitro. We are also determining the role of IL-1 induced 26kD TNF protein in mediating IL-1 induced inhibition of cell growth, and, using retrovirally transfected cells with 26kD and 17kD constructs, we are studying the regulation and kinetics of TNF protein in hormone-dependent and hormone-independent breast cancer cells.